Abstract--Atlantic menhaden (Brevoortia tyrannus), through
landings, support one of the largest commercial fisheries in the United
States. Recent consolidation of the once coast-wide reduction fishery to
waters within and around Chesapeake Bay has raised concerns over the
possibility of the loss of unique genetic variation resulting from
concentrated fishing pressure. To address this question, we surveyed
variation at the mitochondrial cytochrome c oxidase subunit I (COI) gene
region and seven nuclear microsatellite loci to evaluate stock structure
of Atlantic menhaden. Samples were collected from up to three cohorts of
Atlantic menhaden at four geographic locations along the U.S. Atlantic
coast in 2006 and 2007, and from the closely related Gulf menhaden (B.
patronus) in the Gulf of Mexico. Genetic divergence between Atlantic
menhaden and Gulf menhaden, based on the COI gene region sequences and
microsatellite loci, was more characteristic of conspecific populations
than separate species. Hierarchical analyses of molecular variance
indicated a homogeneous distribution of genetic variation within
Atlantic menhaden. No significant variation was found between
young-of-the-year menhaden (YOY) collected early and late in the season
within Chesapeake Bay, between young-of-the-year and yearling menhaden
collected in the Chesapeake Bay during the same year, between YOY and
yearling menhaden taken in Chesapeake Bay in successive years, or among
combined YOY and yearling Atlantic menhaden collected in both years from
the four geographic locations. The genetic connectivity between the
regional collections indicates that the concentration of fishing
pressure in and around Chesapeake Bay will not result in a significant
loss of unique genetic variation.

**********

Atlantic menhaden (Brevoortia tyrannus) is an ecologically and
economically important species along the U.S. East Coast. As a
filter-feeder and key prey fish, it provides a critical link between
primary production and larger piscivorous predators, such as striped
bass (Morone saxatilis), bluefish (Pomatomus saltatrix), and weakfish
(Cynoscion regalis). The commercial fishery for Atlantic menhaden
consists of a small bait fishery and a larger reduction fishery. Of the
20 menhaden reduction plants (where menhaden are "reduced" to
meal and oil) that were once operating along the U.S. Atlantic coast,
only the Reedville, Virginia, facility is currently active. The
concentration of fishing effort for Atlantic menhaden in and around
Chesapeake Bay has raised concerns among many environmentalists and
sport fishermen about the possibility of "localized depletion"
of Atlantic menhaden in the area. A potential consequence of localized
depletion could be the loss of unique genetic variation within Atlantic
menhaden, if there is spatial partitioning of genetic variation (stock
structure) within the species.

Results of previous analyses of the stock structure of Atlantic
menhaden have been discordant. Two populations of Atlantic menhaden, one
north and the other south of Long Island, New York, were suggested on
the basis of vertebral counts and transferrin allele frequencies
(Sutherland, 1963; Epperly, 1989). Two populations, one north and one
south of Cape Hatteras, North Carolina, have also been proposed. This
division was based on the presence of small, sexually mature fish before
the arrival of the larger, migrating fish in North Carolina waters and
the presence of spawning fish off northern Florida in late winter and
early spring (June and Nicholson, 1964). One coast-wide population has
been indicated by tag recovery studies (Nicholson, 1978), which have
shown that Atlantic menhaden of differing ages and sizes share the
over-wintering grounds off Cape Hatteras and undergo seasonal migrations
and that larger fish travel farther north. The Atlantic States Marine
Fisheries Commission currently assesses Atlantic menhaden as a single
coast-wide stock.

In addition to the uncertainty regarding the stock structure of
Atlantic menhaden, the relationship between Atlantic menhaden and Gulf
menhaden (B. patronus) is not well understood. The mean values of
several of the morphometric and meristic characters are significantly
different between the two putative species, although the ranges of
variation are coincident (Dahlberg, 1970). Similarly, preliminary
genetic analyses indicate limited divergence between the putative
species, and therefore the use of larger sample sizes and additional
genetic characters have been recommended (Avise et al., 1989; Anderson,
2007).

In this study, analyses of sequence data from the mitochondrial
cytochrome c oxidase subunit I (COI) gene region and allele frequencies
of seven nuclear microsatellite loci were used to investigate the
genetic relationships of Atlantic and Gulf menhaden, the stock structure
of Atlantic menhaden, and to evaluate the potential for loss of unique
genetic variation resulting from "localized depletion" of
Atlantic menhaden within the Chesapeake Bay region.

[FIGURE 1 OMITTED]

Materials and methods

Sample collection

Young-of-the-year (YOY) and yearling Atlantic menhaden were sampled
from New England (MA), the U.S. mid-Atlantic (NJ), Chesapeake Bay (VA),
and the U.S. South Atlantic (SC) in 2006 and 2007, and YOY Gulf menhaden
were sampled from the Gulf of Mexico in 2006 and 2007 (Table 1; Fig. 1).
For all collections outside the Chesapeake Bay, samples of YOY and
yearling Atlantic menhaden were pooled. For Chesapeake Bay collections,
scales were aged from a sub-sample of 20% of individuals taken in 2007
and length was used as a surrogate for the remaining samples (where fish
less than 100 mm fork length were considered YOY and fish greater than
100 mm fork length were considered yearling). The younger cohorts were
sampled because they are less likely to have migrated far from where
they were spawned. Local experts identified menhaden on the basis of
morphological characters and capture location. Muscle tissue samples
were taken from each individual and either frozen or stored in DMSO
buffer (Seutin et al., 1991) at room temperature. Voucher specimens were
retained from all U.S. Atlantic coast regions in 2007 to corroborate
field identifications. Some of the identifications were retroactively
verified with sequences from a portion of the mtDNA control region,
namely haplotypes were compared to those of Anderson (2007).

Microsatellite loci were amplified by PCR by using locus-specific
fluorescent labels with the conditions outlined in Lynch (2008).
Following amplification, 1 [micro]L of PCR product for each locus was
combined with PCR products from three other unique locus and fluorescent
label combinations (4 [micro]L total), 6 [micro]L HiDi formamide
(Applied Biosystems), and 0.3 [micro]L 500 Liz Gene Scan Size standard
(Applied Biosystems). The reaction mixture was denatured at 95[degrees]C
for 10 minutes before being separated on a 36-cm capillary ABI PRISM[R]
3130x/ Genetic Analyzer (Applied Biosystems) according to the
manufacturer's protocol. The chromatic peaks for each
microsatellite locus were scored by GeneMarker AFLP/Genotyping Software,
vers. 1.60 (SoftGenetics, State College, PA). Once scored, MicroChecker
2.2.3 (Van Oosterhout et al., 2004) was used to check for the presence
of null alleles and evidence of scoring errors. To ensure consistency,
20% of the samples were re-analyzed from the point of DNA extraction
through allele scoring.

Genetic analyses

Once aligned, the mitochondrial sequences were characterized in
Arlequin 3.11 (Excoffier et al., 2005) to determine the number of
haplotypes ([N.sub.h]), number of polymorphic sites (S), and variable
base-pair (bp) locations within a sequence set. Diversity indices,
including haplotype diversity (h), nucleotide sequence diversity ([pi]),
and mean number of pairwise differences (k) within each collection were
also estimated in Arlequin 3.11 (Excoffier et al., 2005). To visualize
genetic relationships among mitochondrial sequences, median-joining
networks were drawn in Network 4.2.0.1 (Bandelt et al., 1999).

For the microsatellite data, Genepop 3.4 (Raymond and Rousset,
1995) was used to determine observed heterozygosity (Ho) and expected
heterozygosity ([H.sub.E]) and to perform exact tests for deviations of
genotypic distributions from the expectations of Hardy-Weinberg
equilibrium for each locus at each collection location (10,000
iterations; Guo and Thompson, 1992). Significance levels were adjusted
for multiple testing by using a Bonferroni correction (Rice, 1989).
Arlequin 3.11 (Excoffier et al., 2005) was used to determine the number
of alleles (a), and Microsatellite Analyzer (MSA) (Dieringer and
SchlStterer, 2003) was used to determine the allele size range (as).
Allelic richness ([R.sub.s]) was estimated in FSTAT 2.9.3.2 (Goudet,
1995).

Using both mitochondrial COI sequence data (OST) and nuclear
microsatellite data ([F.sub.ST]/[R.sub.ST]), we performed a hierarchical
analysis of molecular variance (AMOVA) to test for partitioning of
variation among defined groups. The groups tested were the following:
temporal collections within an age class at a location (e.g., 2007 YOY
in Chesapeake Bay sampled early [May] and late [August] in the season),
between collections of an age class taken at the same location in
different years (e.g., the 2006 year class sampled in Chesapeake Bay as
YOY in 2006 and yearling in 2007), between age classes within a region
(e.g., YOY and yearling menhaden in Chesapeake Bay in 2007), among
Atlantic coast regions both including and excluding the Gulf of Mexico
(e.g., New England, mid-Atlantic, Chesapeake Bay, U.S. South Atlantic,
and Gulf of Mexico), among COI clades (e.g., "Atlantic only,"
"ubiquitous," and "anomalous" samples), and between
Atlantic and Gulf menhaden. AMOVA calculations based on microsatellite
data were analyzed by using both [F.sub.ST] (Weir and Cockerham, 1984)
and [R.sub.ST] (Slatkin, 1995) distance methods. Estimates of population
pairwise [[PHI].sub.ST] and [F.sub.ST]/[R.sub.ST] were calculated in
Arlequin 3.11 (Excoffier et al., 2005) and adjusted for multiple testing
with a Bonferroni correction (Rice, 1989).

To assess the statistical power for detecting population
differentiation with the applied set of microsatellite markers and
sample sizes, a simulation was implemented with POWSIM (Ryman and Palm,
2006), which estimates statistical power by testing different
combinations of number of samples, sample sizes, number of loci, number
of alleles, and allele frequencies for any hypothetical degree of
differentiation ([F.sub.ST]). To match the number of collection
locations and magnitude of [F.sub.ST] estimates in this study, five
hundred replicates were performed on five populations by using
Fischer's method with the following combinations of effective
population size ([N.sub.e]) and generations of drift before sampling
(t): 10,000: 50, 5000: 25, 1000:5 ([F.sub.ST]=0.0025); 10,000: 201,
5000: 100.5, 1000:20.1 ([F.sub.ST]=0.01); 10,000: 1025.8, 5000: 512.9,
1000:102.6 ([F.sub.ST]=0.05). The hypothetical sample sizes were set to
the average across all loci for each sampling location. An additional
simulation was performed with t=O, to assess [alpha] (type-I) error. The
estimate of power was reported as the proportion of significant outcomes
(P<0.05).

Results

The COI fragment was sequenced for 289 Atlantic menhaden and 50
Gulf menhaden. Overall, the fragment contained 99 polymorphic sites (97
in Atlantic menhaden): 5 first codon positions, 1 second codon position,
and 91 third codon positions; 101 transitions (99 in Atlantic menhaden),
7 transversions (6 in Atlantic menhaden); and produced 124 haplotypes
(109 in Atlantic menhaden) (Table 2). All substitutions were synonymous,
resulting in identical amino acid sequences. Haplotype diversity (h)
estimates for the Atlantic and Gulf menhaden sampling locations ranged
from 0.879 in Gulf menhaden to 0.956 in the U.S. mid-Atlantic, with an
overall (pooled) estimate of 0.940 (0.941 for Atlantic menhaden). Mean
nucleotide sequence diversity ([pi]) estimates for Atlantic and Gulf
menhaden sampling locations ranged from 0.0071 in Gulf menhaden to 0.030
in the U.S. South Atlantic, with an overall (pooled) estimate of 0.026
(0.027 for Atlantic menhaden). The mean number of pairwise differences
([kappa]) ranged from 3.2 in Gulf menhaden to 13.5 in the U.S. South
Atlantic, with an overall (pooled) estimate of 12.6 for Atlantic
menhaden and 11.8 for Atlantic and Gulf menhaden combined.

The median-joining network for the 109 COI Atlantic menhaden
haplotypes showed two extensive clusters (clades) separated by 17
substitutions and one minor grouping of three individuals separated by
24 substitutions (Fig. 2). In order to discriminate between the
alternate possibilities that either the three individuals were one of
the other North American Brevoortia species that had been misidentified
in the field as Atlantic menhaden, or that there is incomplete lineage
sorting at the COI locus, the more rapidly evolving mitochondrial
control region was sequenced and compared to those generated by Anderson
(2007) for the four species of North American menhaden (GenBank
accession numbers EF119342-EFl19454). The control region sequences for
the three individuals unambiguously clustered with the "ubiquitous
large-scaled" menhadens in an unweighted pair group method with
arithmetic mean (UPGMA) tree with these sequences, indicating that the
field identifications were correct and that the result was likely
attributable to incomplete lineage sorting at the COI locus. Examination
of the microsatellite genotypes of these three individuals supported
this conclusion.

The distribution of the two major COI clades differed among
Atlantic and Gulf menhaden. Clade I, the "ubiquitous" clade,
comprised all Gulf menhaden samples and 64% of Atlantic menhaden
samples. Clade II, the "Atlantic-only" clade, comprised 35% of
Atlantic menhaden samples and was not detected in Gulf menhaden (Table
2). A contingency table of the four Atlantic menhaden sampling locations
indicated that clades I and II were homogeneously distributed among the
U.S. Atlantic Coast sampling locations ([chi square]=0.478; [[chi
square].sub.005.3]; P>0.05).

Eight microsatellite loci, Aa16, Asa2, Asa4, Asal6, AsaB020,
AsaD055, AsaC334, and SarBH04, were amplified for the Atlantic menhaden
and Gulf menhaden samples. The genotypic distributions of all loci in
all samples conformed to the expectations of Hardy-Weinberg equilibrium
with the exception of locus Asal6 (Table 3). Both the exact test in
Genepop and the MicroChecker analysis revealed a significant or nearly
significant deficiency of Asal6 heterozygotes in all samples, indicating
the presence of a null allele. Consequently, this locus was not included
in any of the population structure analyses.

[FIGURE 2 OMITTED]

For the seven remaining loci, the number of alleles (a) across all
Atlantic menhaden samples ranged from 8 at Aal6 and AsaC334 to 22 at
AsaB020, and the allelic richness ([R.sub.s]) ranged from 7.98 at Aal6
to 21.8 at AsaB020. Allele size ranges were similar for Atlantic
menhaden and Gulf menhaden across all seven loci. However, average a and
Rs values across all loci were lower in Gulf menhaden as compared to
Atlantic menhaden (Table 3).

AMOVAs of the COI haplotype data and microsatellite genotype data
were performed to evaluate the temporal and spatial partitioning of
genetic variation within Atlantic menhaden (Table 4). No significant
differences were detected between early (May) and late (August)
collections of YOY Atlantic menhaden in Chesapeake Bay in 2007.
Likewise, no differences were detected among year classes of Atlantic
menhaden in the Chesapeake Bay based on a comparison of YOY and yearling
menhaden collected in Chesapeake Bay in 2006 or a comparison of YOY and
yearling menhaden collected in Chesapeake Bay in 2007. Following the
same cohort in the same location across years, a comparison of YOY
menhaden collected in 2006 with yearling menhaden collected in 2007 in
the Chesapeake Bay, did not result in a significant difference based on
either the mitochondrial ([[PHI].sub.ST) or the microsatellite
([R.sub.ST]). However, the microsatellite ([F.sub.ST]) AMOVA produced a
significant result, where 1.80% (P=0.02) of the variance was attributed
to differences within the same cohort in Chesapeake Bay in successive
years.

Samples of YOY and yearling menhaden (combined) from four
geographic regions along the U.S. Atlantic Coast (New England,
mid-Atlantic, Chesapeake Bay, and U.S. South Atlantic) were compared to
test for evidence of spatial partitioning of genetic variation. Only the
AMOVA based on the microsatellite [F.sub.ST] was significant,
attributing 0.58% (P<0.001) of variation to sampling location. No
pairwise comparisons of [[PHI].sub.ST], [F.sub.ST] or [R.sub.ST]
revealed statistically significant variation between any two sampling
regions of Atlantic menhaden after a Bonferroni correction (Table 5).

All pairwise comparisons between Atlantic menhaden and Gulf
menhaden collections revealed statistically significant variation. The
mitochondrial ([[PHI].sub.ST]) AMOVA between Atlantic and Gulf menhaden
attributed 18.2% (P<0.001) of the variance to differences between
putative species. Likewise, in the microsatellite comparison, the
[F.sub.ST] and [R.sub.ST] AMOVAs attributed 11.5% (P<0.001) and
38.75% (P<0.001) of variance to variation between Gulf and Atlantic
menhaden samples.

The POWSIM analysis showed that 94.2%, 93.8%, and 92.6% of the
tests where the [N.sub.e]:t combination led to [F.sub.ST]=0.0025
(10,000: 50, 5000: 25, 1000: 5, respectively), 100% of the tests where
the [N.sub.e]:t combination led to [F.sub.ST]=0.01 (10,000: 201, 5000:
100.5, 1000: 20.1), and 100% of the tests where the [N.sub.e]:t
combination led to [F.sub.ST]=0.05 (10,000: 1025.8, 5000: 512.9, 1000:
102.6) were statistically significant, indicating that there was
sufficient statistical power to detect population differences with the
set of microsatellite markers and sample sizes used in this study.

Discussion

The mitochondrial and nuclear markers employed in this analysis
revealed considerable variation within Atlantic menhaden. The seven
microsatellite loci surveyed were highly variable and the number of
alleles per locus and average heterozygosities (a=5-21,
[H.sub.exp]=0.435-0.924) were within the range of variation reported for
other clupeids (a=1-56, [H.sub.exp]=0.066-0.98, Brown et al., 2000;
McPherson et al., 2001; Olsen et al., 2002; Faria et al., 2004; Anderson
and McDonald, 2007; Volk et al., 2007). Typically, one would not
consider using the mitochondrial COI gene region for an analysis of
stock structure because this locus tends to be highly conserved in most
organisms, exhibiting low levels of intraspecific variation (Meyer,
1993). However, previous studies have documented very high levels of
variation throughout the Atlantic menhaden mitochondrial genome (Avise
et al., 1989; Bowen and Avise, 1990; Anderson, 2007), and in a
preliminary analysis of various menhaden mitochondrial gene regions, we
found COI to be sufficiently variable for an analysis of stock
structure. The COI genetic diversity in Atlantic menhaden ([pi]=2.74%)
is an order of magnitude higher than the average within-species
divergence reported for other fishes. For example, Ward et al. (2005)
reported an average z of 0.39% for Australian marine fishes, and Hubert
et al. (2008) reported a [pi] of 0.302% for Canadian freshwater fishes.

There were significant differences in the distribution of COI
haploytpes and microsatellite allele frequencies between Atlantic and
Gulf menhaden, although both classes of markers indicate the two species
are very closely related ([F.sub.ST]=0.104). These results are
consistent with those of Anderson (2007) who surveyed variation at four
microsatellite loci, estimating an [F.sub.ST] of 0.110 between Atlantic
and Gulf menhaden. These [F.sub.ST] values are more typical of
differences between populations than species. For comparison, [F.sub.ST]
values between genetically distinct stocks of clupeid fishes based on
microsatellites range from 0.002 to 0.226 (Shaw et al., 1999; Sugaya et
al., 2008), and are approximately one-fourth of the [F.sub.ST] values
between other pairs of North American menhadens (0.355-0.488; Anderson,
2007).

A low level of genetic divergence between Atlantic and Gulf
menhaden was also noted for the mitochondrial COI gene region
([[PHI].sub.ST]=0.178)--a result consistent with a previous restriction
fragment length polymorphism (RFLP) analysis of the mitochondrial genome
(Avise et al., 1989) and sequence analysis of the control region
(Anderson, 2007) of these two species. In the present study, we did not
find a single COI nucleotide position that distinguished Atlantic from
Gulf menhaden. In a survey of 207 fishes, including several congeners,
Ward et al. (2005) reported that all had different COI sequences.
Furthermore, mean nucleotide differences between closely related species
were 25 times higher (on average) than differences within species. In
the present study, however, the nucleotide sequence diversity for
Atlantic and Gulf menhaden combined ([pi]=0.0258) was less than that for
Atlantic menhaden alone ([pi]=0.0274).

When compared with Gulf menhaden, Atlantic menhaden are generally
larger, have a less convex body shape, and have a higher number of
predorsal scales, vertebrae, and ventral scutes (Bigelow et al., 1963).
Although the mean values of some of the morphometric and meristic
characters are significantly different between the two species, the
ranges of variation are coincident (Dahlberg, 1970). Although Atlantic
menhaden and Gulf menhaden are morphologically similar, their geographic
ranges are not believed to overlap (Bigelow et al., 1963). Thus, the
species are typically distinguished by capture location. The high level
of genetic and morphological similarity of Atlantic and Gulf menhaden
raises concern over the validity of the two species. A thorough
morphological and genetic analysis of the same individuals will be
required to resolve this problem.

In the present analysis of Atlantic and Gulf menhaden mitochondrial
COI gene region sequences, two distinct mitochondrial clades were noted,
one of which was found only in Atlantic menhaden, and the other in both
Atlantic and Gulf menhaden. These results are similar to those found in
a previous RFLP analysis of the whole mitochondrial genome (Arise et
al., 1989) and in a sequence analysis of the control region (Anderson,
2007). Avise (1992) hypothesized that the separation of two
mitochondrial clades between the Atlantic Ocean and the Gulf of Mexico
was a result of historical isolation of Atlantic and Gulf menhaden by
the Florida peninsula during times of cooler water temperatures and
subsequent unidirectional gene flow during geologically recent times.
Anderson (2007) postulated that the distribution of these two clades in
Atlantic menhaden supported very recent gene flow between Atlantic and
Gulf menhaden because the highest frequency of "Atlantic-only"
haplotypes occurred in the northernmost Atlantic menhaden sampling
location. However, the purported geographic cline in the distribution of
the "Atlantic-only" clade haplotypes was only qualitatively
addressed and was based on a small sample size (n=37) of Atlantic
menhaden. In the present study, the more extensive sampling regime for
Atlantic menhaden along the U.S. Atlantic coast (n=289) refutes
Anderson's (2007) hypothesis, because a chi-square analysis of the
presence of the two clades among Atlantic coast sampling locations did
not indicate a heterogeneous distribution.

Population structure

Stock structure analyses of Atlantic menhaden along the U.S.
Atlantic coast have indicated as few as one and as many as three
different stocks based on spawning time, spawning location, and
migration tracks (Sutherland, 1963; June and Nicholson, 1964; Nicholson,
1978; Epperly, 1989). We analyzed the distribution of allelic variation
of rapidly evolving molecular characters to evaluate population
structure of Atlantic menhaden. The resulting AMOVAs did not reveal any
significant portion of molecular variance was due to variation between
the following group comparisons: YOY menhaden collected in Chesapeake
Bay early and late in the season during the same year; YOY and yearling
menhaden collected in Chesapeake Bay in successive years (following the
2006 year class); YOY and yearling menhaden collected in Chesapeake Bay
in the same year (comparing 20052006, 2006-2007 year classes); and YOY
and yearling menhaden (combined) from the four geographic regions along
the U.S. Atlantic coast (New England, mid-Atlantic, Chesapeake Bay, and
U.S. South Atlantic). The POWSIM analysis indicates that the sample
sizes and suite of microsatellite markers used in this study were
sufficient for detecting even weak levels of differentiation ([F.sub.ST]
[greater than or equal to] 0.0025).

Although none of the five COI [[PHI].sub.ST] AMOVAs or five
microsatellite [R.sub.ST] AMOVAs were significant, two of the five
microsatellite [F.sub.ST] AMOVAs showed a small but statistically
significant partitioning of genetic variation between YOY and yearling
menhaden collected in Chesapeake Bay in successive years (following the
2006 year class, 1.80%, P=0.0176) and YOY and yearling menhaden
(combined) from the four geographic regions along the U.S. Atlantic
coast (0.575%, P=0.0000).

The pairwise comparisons between sample locations corroborate the
[[PHI].sub.ST] and [R.sub.ST] AMOVA results. No pairwise comparison
revealed a statistically significant difference between any two of the
four geographic regions of Atlantic menhaden after Bonferroni
correction. These findings support the hypothesis that the significant
results from the [F.sub.ST] AMOVAs were a result of random processes and
not biologically meaningful (for a discussion see Waples, 1998). The
collective results indicate no significant partitioning of genetic
variation between the sampling regions of Atlantic menhaden, and the
null hypothesis that Atlantic menhaden comprise a single stock along the
U.S. Atlantic coast cannot be rejected.

The lack of statistically significant genetic differences among
Atlantic menhaden sampling regions is consistent with the life history
traits of the species. Of all the North American Brevoortia, Atlantic
menhaden undertake the longest coastal migrations and have the most
temporally and geographically protracted spawning season (Whitehead,
1985). Atlantic menhaden are batch spawners, spawning multiple times
during a year. Additionally, Atlantic menhaden larvae are found in
waters from Maine to Mexico and are the most widely distributed larvae
of any clupeoid in the western North Atlantic; (Kendall and Reintjes,
1975). The larvae can take up to 90 days to cross the continental shelf
and are affected by along-shore transport, coastal storms, freshwater
discharge from estuaries, and wind-forcing (Checkley et al., 1988).
Menhaden also undergo an ontogenetic shift in migration, where larger
fish migrate farthest north (Dryfoos et al., 1973). These
characteristics appear to keep Atlantic menhaden--and their gene
pool--well mixed.

Population structure has not been found in genetic analyses of
other clupeids including Atlantic herring (Clupea harengus) (Grant,
1984), twaite shad (Alosa fallax) (Volk et al., 2007), and European
pilchard (Sardina pilchardus) (Gonzalez and Zardoya, 2007). In contrast,
some clupeid species exhibit significant stock structure, often
attributed to the presence of geographic barriers or temporal
reproductive isolation. These include Pacific herring (Clupea pallasi)
stocks in the eastern North Pacific and Bering Sea (Grant and Utter,
1984), in the Bering Sea and Gulf of Alaska separated by the Alaska
Peninsula (O'Connell et al., 1998), and from Honshu and Hokkaido
Islands (Sugaya et al., 2008). Shaw et al. (1999) also found significant
genetic structuring between Icelandic summer-spawners, Norwegian
spring-spawners, and Norwegian fjord stocks of Atlantic herring.

Implications for management

Loss of unique genetic variation due to fishing pressure, habitat
degradation, and hatchery stocking has been reported for Pacific cod
(Gadus macrocephalus), leopard darter (Percina panterina), Japanese
flounder (Paralichthys olivaceus), and American shad (Alosa sapidissima)
(Grant and Stahl, 1988; Echelle et al., 1999; Brown et al., 2000; Sekino
et al., 2003), and there is concern that a concentration of fishing
effort in and around Chesapeake Bay could result in the loss of unique
genetic variation in Atlantic menhaden. In this study we have
demonstrated high genetic variability and a homogeneous distribution of
genetic variation within Atlantic menhaden from four sampling locations
along the U.S. Atlantic coast--a result consistent with the current
management practice that recognizes a single stock of Atlantic menhaden.
The apparent genetic connectivity between New England, mid-Atlantic,
Chesapeake Bay, and U.S. South Atlantic samples indicates that loss of
unique genetic variation due to the consolidation of fishing pressure in
Chesapeake Bay is not likely. Our analysis of mitochondrial and nuclear
loci revealed significant allele frequency differences between Atlantic
and Gulf menhaden, supporting independent management of these resources.
However, the small magnitude of these differences found in this and
previous studies would indicate that a reevaluation of the specific
status of the two putative species, based on analyses of morphological
and genetic characters, is warranted.